The long-term objectives of this research program are the elucidation of the enzymatic mechanisms of steroid transformmtions in animal and microbial systems by the isolation and characterization of the pertinent enzymes, and the application of these basic studies in enzymology and steroid biochemistry to problems of prostatic disease, reproductive function, and inflammation. The immediate specific aims are: (1) To broaden the scope of enzymatic methods for the microestimation of steroids by (a) isolation of novel hydroxysteroid dehydrogenases with more stringent specificities, and (b) simplification of assay procedures through the development of a transhydrogenase assay between NAD and NAD analogs in which steroids act catalytically. (2) Use of these techniques to analyze: (a) Regulation by steroids and gonadotropins of androgen biosynthesis in rat testicular cell cultures by measurement of secreted steroids and steroid-transforming enzymes; and (b) steroid patterns in normal and hyperplastic prostate glands of dogs and men, in the hope of understanding the pathogenesis of the hyperplastic state (BPH) which commonly afflicts man. (3) Elucidation of the properties and functions of the liver 3Alpha-hydroxysteroid dehydrogenase, a major xenobiotic inactivating enzyme, which is also inhibited by steroidal and non-steroidal anti-inflammatory agents. Search for further evidence to show whether this enzyme participates in the mediation of inflammation, by determining its substrate and inhibitor specificities, and its possible role in prostaglandin metabolism. This information may not only shed light on the chemistry of inflammation, but also could provide leads for its control. (4) Description of the detailed molecular structure and catalytic mechanism of the crystalline Delta5-3-ketosteroid isomerase of Pseudomonas testosteroni by NMR, EPR and X-ray diffraction spectroscopy, to delineate the molecular basis for the high specificity and affinity of the steroid-protein binding, and the mechanism of the remarkable catalytic activity of this enzyme. Such information could provide clues to the design of inhibitors of the biosynthesis of steroid hormones, and insight into the fundamental mechanisms of enzyme catalysis.